Bovine Preimplantation Embryos with Silenced Nucleophosmin Mrna Are Able to Develop Until the Blastocyst Stage

REPRODUCTIONRESEARCH

Bovine preimplantation embryos with silenced nucleophosmin mRNA are able to develop until the blastocyst stage

Tereza Toralova´1, Veronika Benesˇova´1,2, Katerˇina Vodicˇkova´ Kepkova´1, Petr Vodicˇka1, Andrej Sˇusˇor3 and Jirˇ´ı Kanˇka1 1Laboratory of Developmental Biology, Institute of Animal Physiology and Genetics Academy of Sciences of the Czech Republic, v.v.i., Rumburska´ 89, 277 21 Libeˇchov, Czech Republic, 2Faculty of Science, Charles University in Prague, Albertov 6, 128 43 Prague, Czech Republic and 3Department of Obstetrics, Gynecology and Reproductive Sciences, Center for Reproductive Sciences, University of California, San Francisco, California 94143-0556, USA Correspondence should be addressed to T Toralova´; Email: [email protected]

Abstract

This study was conducted to investigate the effect of silencing nucleophosmin in the development of in vitro-produced bovine embryos. Nucleophosmin is an abundant multifunctional nucleolar phosphoprotein that participates, for example, in ribosome biogenesis or centrosome duplication control. We showed that although the transcription of embryonic nucleophosmin started already at late eight- cell stage, maternal protein was stored throughout the whole preimplantation development and was sufficient for the progression to the blastocyst stage. At the beginning of embryogenesis, translation occurs on maternally derived ribosomes, the functionally active nucleoli emerge during the fourth cell cycle in bovines. We found that nucleophosmin localisation reflected the nucleolar formation during bovine preimplantation development. The protein was detectable from the beginning of embryonic development. Before embryonic genome activation, it was dispersed throughout the nucleoplasm. The typical nucleolar localisation emerged with the formation of active nucleoli. At the blastocyst stage, nucleophosmin tended to localise especially to the trophectoderm. To see for how long is maternal nucleophosmin preserved, we silenced the nucleophosmin mRNA using RNA interference approach. Although a large portion of nucleophosmin was degraded in embryos with silenced nucleophosmin mRNA, an amount sufficient for normal development was preserved and we detected only a temporal delay in nucleophosmin relocalisation to nucleoli. Moreover, we observed no defects in nuclear shape or cytoskeleton previously found in somatic cells and only a non-significant decrease in embryonic developmental competence. Thus, our results show that the preserved amount of maternal nucleophosmin is sufficient for preimplantation development of bovine embryo. Reproduction (2012) 144 349–359

Introduction biogenesis and centrosome duplication control (Borer et al. 1989, Savkur & Olson 1998, Hingorani et al. The preimplantation development of mammals still 2000, Okuwaki et al. 2002). Moreover, nucleophosmin hides many secrets, especially regarding gene acts as a histone chaperone and is thus involved in expression. After fertilisation, the embryonic genome is controlling chromatin transcription (Swaminathan et al. gradually activated, starting with minor genome activation, presumably initiated at two- to four-cell stages in 2005). Much like many other proteins engaged in bovines and followed by major genome activation at late ribosome biogenesis, nucleophosmin shuttles between eight-cell stage (Memili & First 1998, Kanka 2003). nucleolus and cytoplasm or non-nucleoli region of Although the role of many transcripts during early the nucleus (Borer et al. 1989, Yung et al. 1990, Valdez embryogenesis was revealed (Nganvongpanit et al. et al. 1994, Chen & Huang 2001) and participates in 2006a, 2006b, Toralova et al. 2009, Salilew-Wondim protein transport, for example, of p120 (Valdez et al. et al. 2010) and many potentially important genes were 1994), Rex protein (Adachi et al. 1993), Rev protein identiﬁed (Hamatani et al. 2004, Kanka et al. 2009, (Fankhauser et al. 1991) or nucleolin (Li et al. 1996). Vodickova Kepkova et al. 2011), the role of plenty of At the beginning of mammalian embryogenesis, all the others is still far from clear. mRNAs and plenty of the proteins used by the embryo Nucleophosmin (NPM1, B23, numatrin and NO38) is come from reserves that were generated during oocyte an abundant multifunctional phosphoprotein, whose maturation. Nucleophosmin seems to originate from most important roles are rRNA processing, ribosome maternal reserves at least until the eight-cell stage, when

q 2012 Society for Reproduction and Fertility DOI: 10.1530/REP-12-0033 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via www.reproduction-online.org Downloaded from Bioscientifica.com at 10/02/2021 12:15:35PM via free access 350 T Toralova´ and others it starts to transfer to the developing nucleolus in bovine preimplantation embryo (Svarcova et al. 2007). During the first three cycles, electron-dense spherical masses of tightly packed fibrils, called nucleolar precursor bodies (NPBs), are present in the embryonic nucleus. rRNA synthesis is inactive during this period. Transformation of the NPBs into fibrogranular nucleoli at the time of major genome activation represents the formation of typical rRNA synthesising nucleolus (Camous et al. 1986, King et al. 1988, Kopecny et al. 1989, Pavlok et al. 1993). It has been proposed that nucleophosmin together with nucleolin functions in the assembly of pre-ribosomal particles (Biggiogera et al. 1990). Nucleophosmin inheres in the peripheral portion of the dense fibrillar component and the granular component of nucleolus (Spector et al.1984) that emerges at the end of fourth cell cycle in cattle (Camous et al. 1986, King et al. 1988, Kopecny et al. 1989, Laurincik et al. 2000). Although it seems that maternal nucleophosmin is stored even after the embryonic genome activation (EGA), it is not able to transfer to the evolving nucleoli in a-amanitin-treated embryos and remains dispersed in the nucleoplasm (Bjerregaard et al. Figure 1 Relative abundance of nucleophosmin mRNA during bovine 2004, Svarcova et al. 2007). preimplantation development. The data were normalised according to Silencing of nucleophosmin in somatic cells causes the relative concentration of the external standard (luciferase mRNA, 1 pg per oocyte/embryo). (A) Untreated embryos; (B) embryos treated disorganisation of nuclear and nucleolar structures with with a-amanitin from one-cell stage to four-cell stage and (C) embryos the formation of micronuclei, delays mitosis, suspends treated with a-amanitin from four-cell stage to eight-cell stage. a,b DNA synthesis and induces activation of p53 (Amin et al. Bars show meanGS.D. Values with different superscripts indicate K K 2008a, 2008b). Moreover, many of the NPM1 / cells statistical significance (P!0.05). (MII, MII stage oocyte; 2c, two-cell hold multiple centrosomes or are multinucleated embryo; 4c, four-cell embryo; e8c, early eight-cell embryo; L8c, late (Grisendi et al. 2005). The role of nucleophosmin in eight-cell embryo; mor, morula; bl, blastocyst; 4c AA, four-cell embryo cultivated with a-amanitin; L8c AA, late eight-cell embryo cultivated cell proliferation is still far from clear. Nucleophosmin is with a-amanitin). generally overexpressed in proliferating cells, but on the other hand, it is important for the maintenance of these changes was statistically significant. After EGA, the genome stability and acts as proliferative suppressor mRNA level remained approximately the same until the K K (Grisendi et al. 2005). The Npm1 / mouse embryos blastocyst stage. To see at which stage the transcription were implanted into the endometrium without any of nucleophosmin from embryonic genome is started, detectable defect, nevertheless they die during the embryos were cultured from one-cell stage to four-cell ongoing development (Colombo et al. 2005, Grisendi stage and from four-cell to eight-cell stage in the et al. 2005). Here, we show that even though the amount presence of RNA polymerase II inhibitor a-amanitin at of maternal protein continuously decreases in bovine a final concentration of 100 mg/ml. While we detected no preimplantation embryos, the remaining protein is significant difference in nucleophosmin transcript abun- sufficient for normal preimplantation development of dance in a-amanitin-treated four-cell embryos in embryos with silenced nucleophosmin mRNA. comparison with controls (Fig. 1B), a significant decrease in nucleophosmin mRNA level was found in a-amanitin- treated late eight-cell stage embryos (Fig. 1C). Results The expression of nucleophosmin mRNA from The dynamics of nucleophosmin localisation and embryonic genome starts at late eight-cell stage expression during bovine preimplantation development Quantitative RT-PCR analysis of the expression pattern of As there were some contradictory studies concerning nucleophosmin transcript during in vitro culture (from nucleophosmin localisation during early embryo MII oocyte to blastocyst stage; Fig. 1A) showed a development of cattle (Laurincik et al.2000, Fair significant decrease in transcript level from MII oocyte et al. 2001), we performed the immunofluorescence to two-cell stage. From two-cell stage to early eight-cell analysis of in vitro cultured embryos from two-cell stage stage, the mRNA level slightly decreased and thereafter to blastocyst stage (Fig. 2). In embryos before EGA increased at late eight-cell stage; however, neither of (pre-EGA embryos; two-cell and four-cell embryos),

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Figure 2 Confocal laser scanning microscopy of preimplantation embryos from two-cell stage to hatched blastocyst. In D, D0, and D00 and G, G0, and G00, single nucleus of early eight-cell and morula stage embryo is shown respectively. The embryos were labelled with mouse monoclonal anti-nucleophosmin antibody (A0–I0) and the nuclei were stained with DAPI (A, B, C, D, E, F, G, H, and I). In mergers (A00–I00), nucleophosmin is red and DNA blue. Asterisks in H00 and I00 indicate inner cell mass. nucleophosmin was very abundant and was distributed of nucleophosmin dsRNA efficiently (Fig. 4)and mainly in the nucleoplasm. During mitosis, the protein specifically (Fig. 5) caused degradation of nucleophos- dispersed throughout the whole blastomere with a very min mRNA in bovine preimplantation embryos. slight colocalisation with chromatin. With the formation The nucleophosmin mRNA was reduced by 86.8% of nucleolus, nucleophosmin formed shell-like (P!0.001) in comparison with uninjected control and structures (early eight-cell stage) and consequently, as by 83.6% (P!0.001) in comparison with GFP dsRNA- the nucleolus became functionally active, the local- injected control. No significant difference was found in isation pattern shifted to be typical for somatic cell the abundance of nucleophosmin mRNA between the nucleoli (late eight-cell stage and beyond, i.e. after EGA). uninjected group and GFP dsRNA-injected group We did not detect colocalisation of nucleophosmin and (PO0.05). The experiment was repeated four times. chromatin in post-EGA embryos, and the staining in To verify the specificity of nucleophosmin mRNA mitotic blastomeres was generally much weaker. In late degradation, we measured the level of mRNA of two blastocysts (starting with day 7), nucleophosmin loca- control genes: centromeric protein F (CENPF) and C-type lised predominantly to the trophectoderm (TE). The lectin domain family 2, member D (CLEC2D). We did difference became most apparent in hatched blastocysts. not find any significant difference in mRNA levels Further, we analysed the expression rate in MII between individual groups (PO0.05 in each case). The oocytes, four-cell embryos and morulas using western experiment was repeated four times. blot (Fig. 3B). The amount of nucleophosmin increased Further, we performed the immunoblotting analysis of slightly from MII oocyte to four-cell stage and further four-cell stage embryos and morulas to monitor protein more significant increase was detected at morula stage. silencing. A considerable decrease in protein abundance in nucleophosmin dsRNA-injected embryos compared with uninjected control especially at morula stage was Nucleophosmin mRNA and protein silencing found; however, a small protein amount was still To reveal whether nucleophosmin mRNA expression is detectable (Fig. 6). required for early embryo development, we silenced When we analysed localisation of the residual nucleophosmin mRNA by microinjection of nucleo- nucleophosmin, we found no protein localisation phosmin dsRNA into bovine zygotes. The microinjection changes in embryos injected with nucleophosmin www.reproduction-online.org Reproduction (2012) 144 349–359

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decrease in protein abundance proven by western blot causes some developmental defects or whether the residual protein is sufﬁcient. We did not detect any nucleus shape defect or cytoskeleton deformation (Fig. 9).

Discussion The function of nucleophosmin in somatic cells and the presently available results concerning its expression in Figure 3 (A) The test of anti-nucleophosmin antibody specificity using early embryos suggest its potential importance during western blot analysis. Nucleophosmin lysate was used as positive preimplantation development (Bjerregaard et al. 2004, control and empty vector was used as negative control. Twenty-six Svarcova et al. 2007, Amin et al. 2008a, 2008b). To embryos at the morula stage were analysed at the same time. provide an experimental basis for this assumption, we (B) Quantification of nucleophosmin protein level after western blot determined the level of nucleophosmin mRNA analysis of bovine oocytes and preimplantation embryos (45 embryos per lane). The data were processed using Quantity One software expression in individual developmental stages of early (Bio-Rad). 100% represents the sum of trace quantity of all bands; embryogenesis with special emphasis on the EGA stage. relative abundance (y-axis) represents the percentage of each band. The expression profile of genes in individual stages a,b Bars show meanGS.D. Values with different superscripts indicate reflects their importance during preimplantation statistical significance (P!0.05). The experiment was repeated three development, genes activated immediately at EGA times, and the representative western blot picture is shown below the being assumed to be the most important. As we found graph. (MII, MII oocytes; 4c, four-cell stage embryos). that the embryonic transcription starts from nucleophos- dsRNA when compared with both control groups, min gene at late eight-cell stage, the transcription of whether in embryos arrested in early stages or normally nucleophosmin from embryonic genome seems to be developing embryos (Fig. 7). We only detected a slight necessary and hence we proceeded to further analysis. delay in nucleophosmin relocalisation from nucleo- We wanted to determine the expression and local- plasm to nucleoli. In eight-cell stage, only five of isation pattern of nucleophosmin protein during whole 60 blastomeres (8.3%) in uninjected group and three of preimplantation development. During early embryogen- 53 blastomeres (5.6%) in GFP dsRNA-injected group still esis, nucleophosmin localisation clearly reflected the displayed entirely nucleoplasmic localisation, while formation of the nucleolus. In pre-EGA embryos, where the same was true for 18 of 73 blastomeres (24.6%) in no functional nucleolus is present, the protein was nucleophosmin dsRNA-injected embryos. Nevertheless, dispersed throughout the nucleoplasm, and during mitosis, it diffused all over the blastomere (Fig. 2A, the localisation was purely nucleolar already at the 0 00 0 00 16-cell stage in all three experimental groups. A ,A ,B,B,andB ). In pre-EGA embryos, we were able to detect nucleophosmin staining earlier Laurincik et al. (2000) and Svarcova et al. (2007),whoshow Developmental competence of nucleophosmin dsRNA-injected embryos is not significantly diminished To investigate the effect of nucleophosmin mRNA silencing on preimplantation development, we mon- itored the capacity of embryos in all three experimental groups to develop from two-cell stage to blastocyst stage. We found a decrease in the number of embryos injected with nucleophosmin dsRNA that reached the blastocyst stage. This deterioration was clearly noticeable in comparison to both control groups (meanGS.D.: uninjected control, 31.67%G8.27; GFP dsRNA-injected control, 26.76%G11.50; nucleophosmin dsRNA injected, 13.96%G7.28), but the difference was not statistically significant (A significant, however boundary, P value (PZ0.05) was found when comparing nucleo- Figure 4 Relative abundance of nucleophosmin mRNA after injection phosmin dsRNA-injected group and uninjected group.) of nucleophosmin dsRNA. The relative abundance (y-axis) represents (Fig. 8). The experiment was repeated four times. the amount of nucleophosmin mRNA in a single embryo normalised to K K Of several defects found in NPM1 / cells and the mean of mRNA expression in uninjected embryos in each developmental stage. In total, 15 uninjected, 12 GFP dsRNA-injected embryos (Colombo et al. 2005, Grisendi et al. 2005, and 16 nucleophosmin dsRNA-injected embryos were analysed. Bars a,b Amin et al. 2008a, 2008b), we focused on the defect in shows meanGS.D. Values with different superscripts indicate tubulin polymerisation. We wanted to know whether the statistical significance (P!0.05).

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K K showed that the placental structures in NPM1 / mice embryos are smaller, even though normally developed. Interestingly, Johansson & Simonsson (2010) reported that nucleophosmin forms complexes with ICM markers POU5F1, SOX2 and NANOG in embryonic stem (ES) cells. It is possible that in these protein interactions, one protein silences its binding partner or that nucleophosmin is still needed for ICM development, but low amount is sufﬁcient in this phase. Svarcova et al. (2007) in bovine embryos and Bjerregaard et al.(2004)in porcine embryos reported that maternal nucleophosmin is stored at least over the EGA stage, but it is not able to localise to the nucleoli. However, both used the general transcription inhibitor a-amanitin. Hence, it cannot be distinguished whether maternal nucleophosmin is naturally preserved or whether it is just not degraded due to the absence of another protein. Similarly, it cannot be distinguished whether maternal protein is not able to translocate to the nucleolus or whether this is caused by the absence of some nucleophosmin-translocating protein. Hence, we employed the microinjection of nucleophosmin dsRNA into bovine zygotes, which causes sequence-speciﬁc degradation of nucleophosmin mRNA. This experiment showed that relocalisation of the protein is only tempor- arily delayed at eight-cell stage. However, as soon as at 16-cell stage, it is localised in the same manner as in

Figure 5 The expression of control genes after injection of nucleophosmin dsRNA. The relative abundance of (A) CENPF and (B) CLEC2D. The relative abundance (y-axis) represents the amount of mRNA in a single embryo normalised to the mean of mRNA expression in uninjected embryos in each developmental stage. Bars show meanGS.D. nucleophosmin staining from the four-cell stage, and only in some embryos. These variances can, however, be caused by different culture media or by the usage of different antibodies (Shinmura et al. 2005). As the vacuolised nucleoli are being formed at eight- cell stage, nucleophosmin generates the so-called shell- like structures (Fig. 2C, C0,C00,D,D0, and D00). After EGA, functionally active nucleolus is present from the beginning of the cell cycle and nucleophosmin displays its typical localisation pattern characteristic for somatic cells (Fig. 2E, E0,E00,F,F0, and F00). From day 7 blastocyst, nucleophosmin tended to localise mainly to TE cells and the distinction was apparent especially in hatched Figure 6 Quantiﬁcation of nucleophosmin protein level after western 00 00 blastocysts (Fig. 2H and I ). As nucleophosmin is blot analysis of bovine embryos at four-cell and morula stages overexpressed in rapidly dividing cells and its down- (collection at 44 and 120 hpf respectively) following nucleophosmin regulation suppresses proliferation (Wang et al. 2006, dsRNA injection. The expression level of uninjected four-cell stage G a,b Okuwaki 2008, Qing et al. 2008, Johansson et al. 2010), embryos was considered 100%. Bars show mean S.D. Values with different superscripts indicate statistical signiﬁcance (P!0.05). the elevated expression in TE cells is likely in connection The experiment was repeated two times, the representative western blot with preparation for embryo implantation. Correspond- picture and the loading control using a-tubulin antibody are shown ingly, Colombo et al. (2005) and Grisendi et al. (2005) below the graph. www.reproduction-online.org Reproduction (2012) 144 349–359

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Figure 7 Confocal laser scanning microscopy of normally developing and arrested embryos at morula stage after injection of nucleophosmin dsRNA and corresponding controls. In total, 58 uninjected embryos, 39 GFP dsRNA-injected embryos and 30 nucleophosmin dsRNA-injected embryos were analysed. Nuclei (DAPI) – blue; nucleophosmin – red. control embryos and no localisation differences were starting amount of the protein, which causes the found during consequent embryo development (Fig. 7). decrease in developmental competence. In favour of Further, we observed no nuclear shape or cytoskeleton the protein preservation speaks also the result on murine K K defects reported by Amin et al. (2008a) (Fig. 9) and only NPM1 / mutants (Colombo et al. 2005, Grisendi et al. K K an undistinguished difference in the developmental 2005). The NPM1 / embryos arrest their development competence (Fig. 8). This likely shows that the preserved at around midgestation and until then they likely use the amount of maternal protein is mostly sufﬁcient. pool of nucleophosmin protein synthesised from However, as the mRNA expression in single preimplan- maternal mRNA. tation embryo is very variable (see S.D.barsinFig. 4) and In conclusion, we show that the transcription of the cell number increases, the protein level might nucleophosmin mRNA from embryonic genome is become insufﬁcient for some embryos with lower activated at late eight-cell stage. The protein is present

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penicillin, 50 mg/ml streptomycin, 10% oestrus cow serum (ECS) and gonadotropins (P.G. 600, 15 U/ml; Intervet, Boxmeer, Holland) without paraffin overlay in four-well dishes under humidified atmosphere for 24 h at 39 8C with 5% CO2. For IVF, the cumulus–oocyte complexes were washed four times in PBS and once in fertilisation medium Tyrode’s albumin lactate pyruvate (TALP) and transferred in groups of up to 30 into four-well dishes containing 250 ml TALP per well. The TALP medium contained 1.5 mg/ml BSA, 30 mg/ml heparin, 0.25 mM sodium pyruvate, 10 mM lactate and 20 mM penicillamine. One straw with frozen sperm of one bull previously tested in the IVF system was thawed in 40 8C water bath, diluted with 2 ml TALP and centrifuged at 3500 g for 10 min. The spermatozoa were layered under 5!1 ml TALP. Figure 8 Developmental competence of embryos after injection of The supernatant with the motile spermatozoa was isolated nucleophosmin dsRNA. Number of embryos reaching individual after 40 min of swim-up at 39 8C(Pavlok et al.1992). developmental stages (y-axis). The number of two-cell stage embryos is Spermatozoa were counted in a haemocytometer and diluted considered as 100%. The developmental competence was followed up in the appropriate volume of TALP to give a concentration of during four independent experiments; in total, 77 uninjected, 68 GFP 2!106 spermatozoa/ml. A 250 ml aliquot of this suspension dsRNA injected and 65 nucleophosmin dsRNA injected two-cell stage embryos. a,bValues with different superscripts indicate PZ0.05. 2c, was added to each fertilisation well to obtain a final con- ! 6 two-cell stage embryos; 4c, four-cell stage embryos; 8c, eight-cell stage centration of 1 10 spermatozoa/ml. Plates were incubated embryos; 16c, 16-cell stage embryos. under humidified atmosphere with 5% CO2 for 20 h at 39 8C. Following fertilisation, presumed zygotes were denuded by from the beginning of embryonic development and its gentle pipetting and transferred to EmbryoAssist medium localisation reflects the formation of the nucleoli. (Origio, Jyllinge, Denmark) supplemented with 10% ECS and A small amount of maternal protein is preserved cultured in humidified atmosphere of 5% CO2–5% O2–90% throughout the whole preimplantation development N2 (25 zygotes in 25 ml medium under liquid paraffin; Origio). and enables almost normal growth of embryos with At morula stage, the EmbryoAssist medium was replaced by silenced nucleophosmin mRNA. BlastAssist medium and embryos were cultivated till hatched blastocyst stage. The dishes were examined at 24 h post- isolation and 32, 44, 56, 92, 104, 120, 156 and 180 h post- Materials and Methods fertilisation (hpf), and MII oocytes and two-cell, four-cell, early eight-cell, late eight-cell embryos, 16-cell embryos, morula, IVF and embryo culture blastocysts and hatched blastocysts were collected at each time Unless otherwise indicated, the chemicals were purchased point respectively. from Sigma (Sigma–Aldrich) and plastic from Nunclon (Nunc, Roskilde, Denmark). Bovine embryos were obtained after Quantification of mRNA expression in individual in vitro maturation of oocytes and their subsequent fertilisation developmental stages and in vitro culture. Briefly, abattoir-derived ovaries from cows and heifers were collected and transported in thermocontainers Poly (A)C mRNA was extracted from the pools of 20 oocytes in sterile saline at about 33 8C. The follicles with diameter and embryos in each stage of development, using a Dynabeads between 5 and 10 mm were dissected with fine scissors and mRNA DIRECT Micro Kit (Invitrogen Dynal AS) according to then punctured. The cumulus–oocyte complexes were eval- the manufacturer’s instructions. Before isolation, 1 pg Lucifer- uated and selected according to the morphology of cumulus ase mRNA (Promega) per oocyte/embryo was added as an and submitted to in vitro maturation in TCM 199 (Earle’s salt) external standard. Primer sequences were designed using supplemented with 20 mM sodium pyruvate, 50 U/ml Beacon Designer 7 from bovine nucleophosmin gene

Figure 9 Confocal laser scanning microscopy of eight-cell stage embryos injected with nucleophosmin dsRNA and corresponding controls stained for nucleophosmin and a-tubulin as a cytoskeletal protein. In total, 15 uninjected, eight GFP dsRNA- injected and 15 nucleophosmin dsRNA-injected embryos were analysed. Nuclei (DAPI) – blue; nucleophosmin – red; a-tubulin – green. www.reproduction-online.org Reproduction (2012) 144 349–359

Downloaded from Bioscientifica.com at 10/02/2021 12:15:35PM via free access 356 T Toralova´ and others sequence (GenBank accession number NM_001035441; stored at K80 8C. Control embryos were collected at the same Table 1). The expression of specific mRNA was measured by time interval as their treated counterparts from the same quantitative RT-PCR. mRNA equivalent of one embryo was fertilisation/cultivation group, washed with PBS, immediately amplified by a OneStep RT-PCR kit (Qiagen) with real-time frozen and stored at K80 8C. All pools were done in triplicate detection using SybrGreenI fluorescent dye. Reaction compo- and contained 20 embryos. sition was QIAGEN OneStep RT-PCR Buffer (1!), dNTP Mix (400 mM of each), forward and reverse primers (both 400 mM), SybrGreenI (1:50 000 of 1000! stock solution; Invitrogen), Synthesis of dsRNA RNasin Ribonuclease Inhibitor (Promega; 0.2 ml), QIAGEN The RNA for nucleophosmin DNA template synthesis was OneStep RT-PCR Enzyme Mix (0.5 ml) and template RNA. isolated from bovine fibroblasts using RNeasy Mini Kit Reaction conditions were as follows: RT at 50 8C for 30 min, (Qiagen). The template was synthesised using primers ‘NPM1 initial activation at 95 8C for 15 min, cycling: denaturation dsRNA’ (see Table 1). The identity of fibroblastic and embryonic at 94 8C for 15 s, annealing at 53 8C for 20 s and extension at sequence was verified by sequencing. These primers generated 72 8C for 30 s. The final extension step was held for 10 min amplicons corresponding to the bovine cDNA sequence in at 72 8C. The real-time RT-PCRs were run in duplicates, with all GenBank (NM_001035441.1). The green fluorescent protein samples (oocytes and all embryo stages) in the same reaction. (GFP) DNA template was amplified from empty p-Bluescript- The experiments were carried out on RotorGene 3000 (Corbett GFP vector (kindly donated by M Anger and P Sˇolc) using Research, Mortlake, NSW, Australia/Qiagen). Fluorescence primers ‘GFP dsRNA’ (Anger et al. 2005; see Table 1). Both data were acquired at 3 8C below the melting temperature to pairs of primers were fused with the T7 promoter. distinguish the possible primer dimers. The RT and the PCRs were performed using two-step Phusion The relative concentration of template in different samples RT-PCR kit (Finnzymes, Vantaa, Finland) for nucleophosmin was determined using comparative quantification in analysis template synthesis; the sole PCR was performed using Phusion software (Corbett Research/Qiagen) as described in Kanka et al. Hot Start II DNA Polymerase (Finnzymes) for GFP DNA (2009). The results were normalised according to the relative template synthesis. The RT was performed with oligo(dT) concentration of the external standard (Luciferase). The take-off primers and reaction conditions were primer extension at points were calculated as 20% of the second-derivative 25 8C for 10 min, cDNA synthesis at 50 8C for 60 min and maximum level (RotorGene 3000 operation manual; Corbett reaction termination at 85 8C for 5 min. Reaction composition Research). Products were verified by melting analysis and gel for PCR was 5! Phusion HF Buffer, dNTP Mix (600 mMof electrophoresis on 1.5% agarose gel with ethidium bromide each), forward and reverse primers (both 400 mM), Phusion staining. Experiment was repeated three times. Hot Start DNA Polymerase (0.15 ml) and template RNA. The reaction conditions for PCR were initial denaturation at 98 8C for 3 min cycling and denaturation at 98 8C for 10 s, annealing a-Amanitin treatment at 55 8C for 5 s and extension at 72 8C for 30 s. The final To block RNA polymerase II-dependent transcription, extension step was held for 5 min at 72 8C. The PCR product a-amanitin (Sigma–Aldrich) was added to the culture medium was purified using QIAquick PCR Purification Kit (Qiagen) and at a final concentration of 100 mg/ml either from one-cell stage the identity was confirmed by sequencing. to four-cell stage (20–44 hpf) or from four-cell stage to late The DNA template coupled with T7 promoter was eight-cell stage (44–92 hpf). After the a-amanitin treatment, the transcribed in vitro using MEGAscript RNAi Kit (Ambion). embryos were washed with PBS, immediately frozen and An amount of 1 mg DNA template was used for each reaction.

Table 1 Primer details. Annealing Amplicon Primer Sequences temperature (8C) size (bp) NPM1 (NM_001035441) dsRNA 50-AGGATCCTAATACGACTCACTATAGGGAGAACGATGAC- 55 583 GATGATGATGATG-30 50-ACTCGAGTAATACGACTCACTATAGGGAGAACAAG- CAAAGGGTGGAGTTC-30 GFP dsRNAa (Anger et al. 2005)50-AGGATCCTAATACGACTAACTATAGGGAGAATGGTGAG- 55 712 CAAGGGCGAGGA-30 50-ACTCGAGTAATACGACTCACTATAGGGAGAGCGGCCGCTT- TACTTGTACA-30 CLEC2D (XM_869843) 50-CACATGCCACGGAACAGC-30 55 110 50-CTGCGGAGGACAGATTCTTG-30 NPM1 (NM_001035441) 50-ACAGCCAACGGTTTCTCTTG-30 55 154 degradation veriﬁcation 50-TTTCACCTCCTCCTCCTCCT-30 CENPF (XM_612376) 50-AGATGAAAGCCAGGCTCACCCAGGAGCTAC-30 60 445 50-TCCAGGTCAGCCAAGGCAAGCTTCAGTTTC-30 NPM1 (NM_001035441) mRNA 50-CTGCTGGTTCCAATAGTAGTC-30 53 262 expression 50-CGCCTCTGCTTCAACAAC-30 Luciferase 50-ACTTCGAAATGTCCGTTCGG-30 55 633 50-ACTTCGAAATGTCCGTTCGG-30 aTranscribed from empty p-Bluescript-GFP vector; kindly donated by M Anger and P Sˇolc.

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The reaction mixture was incubated for 5 h at 37 8C and the at 94 8C for 20 s, annealing at temperature according to the sense and antisense strands were transcribed in the same primer for 20 s and extension at 72 8C for 30 s. The final reaction. The residual DNA template and ssRNA were digested extension step was held for 10 min at 72 8C. and the dsRNA was purified according to the manufacturer’s The experiments were carried out on RotorGene 3000 instruction. One microlitre RNA acquired by in vitro transcrip- (Corbett Research/Qiagen). Fluorescence data were acquired at tion and 1 ml final dsRNA were resolved by electrophoresis on 3 8C below the melting temperature to distinguish the possible 1.5% agarose gel to confirm the integrity of the dsRNA and primer dimers. The qRT-PCR data were determined using serial efficiency of the annealing step. dilutions and the standard curve was created using the take-off points. The take-off points were calculated by Internal RotorGene software (Corbett Research) and defined as the Zygote microinjection cycle at which the second-derivative curve is at 20% of the The zygotes were injected 20 hpf at the stage of two pronuclei. maximum rate of fluorescence and indicates the transition to dsRNA was dissolved in RNase-free water to a final con- the exponential phase (RotorGene 3000 operation manual; centration of 800 ng/ml. Two control groups were established – Corbett Research). The starting amount of corresponding RNA the uninjected group and a group injected with GFP dsRNA. in analysed samples was determined by appointing the take-off Zygotes were microinjected with w5 pl of the dsRNA using points to the curve. Products were verified by melting analysis an MIS-5000 micromanipulator (Burleigh, Exfo Life Sciences, and gel electrophoresis on 1.5% agarose gel with ethidium Quebec, QC, Canada) and PM2000B microinjector (Micro- bromide staining. Data Instrument, South Plainfield, NJ, USA). Pipettes for microinjection were made using P97 Pipette Puller (Sutter Immunofluorescence Instrument Company, Novato, CA, USA). In total, 19 independent experiments were performed. Nucleophosmin staining Embryos were categorised into the following groups: i) embryos Embryos were fixed in 4% paraformaldehyde supplemented injected with nucleophosmin dsRNA (928 zygotes), ii) embryos with 0.5% (v/v) TritonX-100 for 50 min at 4 8C. Fixed embryos injected with GFP dsRNA (665 zygotes) and iii) uninjected were processed immediately or stored in PBS up to 3 weeks at embryos (795 zygotes). 4 8C. After washing in PBS, the embryos were incubated in 0.5% After microinjection, embryos were cultivated under (v/v) TritonX-100 for 15 min. All subsequent steps were done standard conditions and collected at early eight-cell, late in PBS supplemented with 0.3% (w/v) BSA and 0.05% (w/v) eight-cell, 16-cell, morula or blastocyst stage (see earlier). The saponin (PBS/BSA/sap). Embryos were blocked with 2% (v/v) number of embryos that reached each developmental stage normal goat serum (NGS; Millipore Biosciences; St Charles, was counted and the morphological state of each embryo was MO, USA) for 1 h and incubated with mouse anti-nucleophosmin determined using phase-contrast technique. antibody that reacts with C-terminus of the protein (Invitrogen) 1:100 in PBS/BSA/sap overnight at 4 8C. After thorough washing, Evaluation of nucleophosmin mRNA degradation the embryos were incubated with goat anti-mouse antibody conjugated with Alexa 594 in PBS/BSA/sap for 1 h at room The embryos fixed after microinjection (GFP and nucleophos- temperature in the dark. Controls of immunostaining specificity min dsRNA injected) and the uninjected embryos were washed were carried out by omitting primary antibody or using another using FCW buffer (a component of FastLane Cell SYBR Green species-specific secondary antibody conjugate. Kit; Qiagen) and stored dry and deep-frozen at K80 8C until used. Whole single embryos were lysed in 10 ml of the mixture Nucleophosmin and a-tubulin double staining of FCPL buffer and gDNA Wipeout buffer 2 (both components of FastLane Cell RT-PCR kit; Qiagen) according to the The differences specific for double staining are mentioned manufacturer’s instructions and the lysate was directly used below; remaining steps were done as described earlier. Embryos for the RT-PCR. were fixed in 4% paraformaldehyde for 50 min at 4 8C and Quantitative RT-PCR was performed using OneStep RT-PCR subsequently permeabilised in 0.5% TritonX-100 for 10 min at kit (Qiagen) with real-time detection using SybrGreenI room temperature. Samples were further incubated with mouse fluorescent dye. In addition to nucleophosmin mRNA evalu- anti-nucleophosmin antibody and consequently with goat anti- ation, analysis of CENPF and CLEC2D mRNA expression level mouse antibody conjugated with Alexa 594. From now on, all was performed as control of degradation specificity. Reaction steps were performed in dark. After thorough washing, the composition was QIAGEN OneStep RT-PCR buffer (1!), dNTP embryos were incubated in 2% (v/v) NGS for 1 h and then with Mix (400 mM each), forward and reverse primers (both rabbit anti-a-tubulin antibody (Abcam, Cambridge, UK) 1:500 400 mM), SybrGreenI (1:50 000 of 1000! stock solution; in PBS/BSA/sap overnight at 4 8C. After thorough washing, the Invitrogen), RNasin Ribonuclease Inhibitor (Promega; 0.2 ml), embryos were incubated in goat anti-rabbit antibody con- QIAGEN OneStep RT-PCR Enzyme Mix (0.5 ml) and template jugated with FITC (Santa Cruz Biotechnology, Santa Cruz, CA, RNA. For control of degradation efficiency, ‘NPM1 degradation USA) for 1 h. Controls of immunostaining specificity were verification’ primers were used; for control of degradation carried out by omitting one or both primary antibodies, while specificity, CLEC2D and CENPF primers were used (see using both secondary antibodies. Table 1). Reaction conditions were RT at 50 8C for 30 min, The nuclei were stained and the embryos were mounted on initial activation at 95 8C for 15 min and cycling: denaturation glass slides using VECTASHIELD HardSet Mounting Medium www.reproduction-online.org Reproduction (2012) 144 349–359

Downloaded from Bioscientifica.com at 10/02/2021 12:15:35PM via free access 358 T Toralova´ and others with DAPI (Vector Laboratories, Peterborough, UK). The Acknowledgements samples were examined with Leica TCS SP confocal laser The authors are indebted to J Tyleckova, R Hrabakova, V Baran, scanning microscope (Leica Microsystems AG, Wetzlar, P Karabinova and L Liskova for their helpful comments and Germany). The images were processed using the ImageJ expert assistance during the experiments; to M Kopcikova, software (NIH, Bethesda, MD; http://rsb.info.nih.gov/ij). J Kankova, J Klucinova, J Sestakova and J Supolikova for the excellent technical assistance and to O Sebesta for his help Western blotting with the confocal microscope. Unless otherwise indicated, chemicals were purchased from Sigma. Embryos and oocytes (45 per extract for uninjected MII, 4c and morulas or 26 per extract for injected 4c and morulas References and corresponding uninjected controls) were lysed in 15 ml1! Blue Loading Buffer (7722, Cell Signaling Technology, Adachi Y, Copeland TD, Hatanaka M & Oroszlan S 1993 Nucleolar targeting signal of Rex protein of human T-cell leukemia virus type I Danvers, MA, USA) with dithiothreitol, boiled for 5 min and specifically binds to nucleolar shuttle protein B-23. Journal of Biological subjected to 12% SDS–PAGE. Proteins were transferred Chemistry 268 13930–13934. from gels to Immobilon P membrane (Millipore Biosciences, Amin MA, Matsunaga S, Uchiyama S & Fukui K 2008a Depletion of Billerica, MA, USA) using a semidry blotting system (Whatman nucleophosmin leads to distortion of nucleolar and nuclear structures in HeLa cells. Biochemical Journal 415 345–351. (doi:10.1042/ Biometra GmbH, Hoettingen, Germany) for 28 min at 2 BJ20081411) 5 mA/cm . The blocking of the membrane was performed in Amin MA, Matsunaga S, Uchiyama S & Fukui K 2008b Nucleophosmin is 5% non-fat milk in TBS–Tween buffer (TBS-T, 20 mM Tris, pH required for chromosome congression, proper mitotic spindle formation, 7.4, 137 mM NaCl and 0.5% Tween 20) for 1 h and incubated and kinetochore-microtubule attachment in HeLa cells. FEBS Letters 582 overnight with mouse anti-nucleophosmin antibody (1:1000) 3839–3844. (doi:10.1016/j.febslet.2008.10.023) Anger M, Stein P & Schultz RM 2005 CDC6 requirement for spindle or anti-a-tubulin antibody (1:2000) in 5% non-fat milk/TBS-T. formation during maturation of mouse oocytes. Biology of Reproduction After washing in TBS-T, the membranes were incubated with 72 188–194. (doi:10.1095/biolreprod.104.035451) HRP-conjugated donkey anti-mouse IgG antibody (1:7500; Biggiogera M, Bu¨rki K, Kaufmann SH, Shaper JH, Gas N, Amalric F & Jackson Immuno Research, Suffolk, UK) in 3% non-fat Fakan S 1990 Nucleolar distribution of proteins B23 and nucleolin in mouse preimplantation embryos as visualized by immunoelectron milk/TBS-T for 1 h at room temperature. Proteins were microscopy. Development 110 1263–1270. visualised by the ECL-PLUS detection system (Amersham Bjerregaard B, Wrenzycki C, Strejcek F, Laurincik J, Holm P, Ochs RL, Biosciences) according to the manufacturer’s instruction. The Rosenkranz C, Callesen H, Rath D, Niemann H et al. 2004 Expression of data were processed using Quantity One software (Bio-Rad). nucleolar-related proteins in porcine preimplantation embryos produced in vivo and in vitro. Biology of Reproduction 70 867–876. (doi:10.1095/ The specificity of the anti-nucleophosmin antibody was biolreprod.103.021683) tested using Nucleophosmin Lysate NBL1-13748 (Novus Borer RA, Lehner CF, Eppenberger HM & Nigg EA 1989 Major nucleolar Biologicals, Littleton, CO, USA). Two hundred microlitres of proteins shuttle between nucleus and cytoplasm. Cell 56 379–390. 1! Blue Loading Buffer were added to 10 ml nucleophosmin (doi:10.1016/0092-8674(89)90241-9) Camous S, Kopecny V & Flećhon JE 1986 Autoradiographic detection of the lysate or empty vector (supplied with nucleophosmin lysate). earliest stage of [3H]-uridine incorporation into the cow embryo. Biology Ten microlitres of the mixture were subjected to 12% SDS– of the Cell 58 195–200. (doi:10.1111/j.1768-322X.1986.tb00506.x) PAGE followed by classic western blot procedure (Fig. 3A). Chen D & Huang S 2001 Nucleolar components involved in ribosome biogenesis cycle between the nucleolus and nucleoplasm in interphase cells. Journal of Cell Biology 153 169–176. 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The authors declare that there is no conflict of interest that Molecular and Cellular Biology 11 2567–2575. Grisendi S, Bernardi R, Rossi M, Cheng K, Khandker L, Manova K & could be perceived as prejudicing the impartiality of the Pandolfi PP 2005 Role of nucleophosmin in embryonic development and research reported. tumorigenesis. Nature 437 147–153. (doi:10.1038/nature03915) Hamatani T, Carter MG, Sharov AA & Ko MSH 2004 Dynamics of global gene expression changes during mouse preimplantation development. Developmental Cell 6 117–131. (doi:10.1016/S1534-5807(03)00373-3) Hingorani K, Szebeni A & Olson MO 2000 Mapping the functional Funding domains of nucleolar protein B23. Journal of Biological Chemistry 275 This work was supported by GACR 523/09/1035; T Toralova´ 24451–24457. (doi:10.1074/jbc.M003278200) Johansson H & Simonsson S 2010 Core transcription factors, Oct4, Sox2 was supported by GACR 204/09/H084 and V Benesˇova´ and and Nanog, individually form complexes with nucleophosmin (Npm1) to T Toralova´ were supported by GAUK 43-251133. control embryonic stem (ES) cell fate determination. Aging 2 815–822.

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